Preparation method of rana temporaria chensinensis collagen peptide

Abstract

The present application belongs to the technical field of extraction and preparation of forest frog collagen peptide, and particularly relates to a method for extracting and preparing forest frog collagen peptide. Fresh and live northeast forest frogs are selected, the whole skin containing attached bones is peeled, non-target tissues such as fascia and fat are removed, and blood stains are removed by washing with clean water. The skin is air-dried to a water content of ≤15% under the condition of a temperature of 25-30°C and a relative humidity of ≤60%, and then screened through a 10-mesh standard sieve and blown to remove dust to obtain clean raw materials. The clean raw materials are ground at high speed and then passed through an 80-mesh standard sieve to obtain raw material powder with a particle size of ≤180 μm. The raw material powder is mixed with 15-20-year-old aged yellow rice wine at a mass ratio of 1:1.2-1.5 to form a uniform suspension system. Neutral protease is added to the suspension system, and enzymolysis is carried out at 48-50°C for 12-20 hours until the change rate of the content of soluble protein in the system is ≤5% / h to obtain an enzymolysis solution. The enzymolysis solution is sequentially filtered through three layers, allowed to stand and precipitate, and filtered through a gradient filter screen to obtain forest frog collagen peptide finished product.

Description

Technical Field

[0001] This invention belongs to the field of extraction and preparation technology of forest frog collagen peptides, and particularly relates to a method for extraction and preparation of forest frog collagen peptides. Background Technology

[0002] Traditional collagen peptide extraction methods often use pig skin and fish skin as raw materials, which suffer from problems such as low raw material utilization, stringent enzymatic hydrolysis conditions, and insufficient product purity. Northeast forest frog skin is rich in natural active collagen, making it an excellent raw material for preparing high-quality collagen peptides. However, existing forest frog collagen extraction processes generally suffer from shortcomings such as crude pretreatment, poor controllability of enzymatic hydrolysis, and incomplete removal of impurities, making it difficult to consistently obtain high-purity and high-activity forest frog collagen peptide products.

[0003] Currently, the preparation of frog collagen peptides mainly adopts a combination of single enzymatic hydrolysis and simple filtration processes, which have defects such as low extraction efficiency, residual large molecular impurities, and unstable product physicochemical indicators. In addition, the process parameters lack precise control, resulting in significant batch-to-batch product quality differences. This cannot meet the core requirements of large-scale production and market application for product purity, safety, and stability. There is an urgent need for an efficient, mild, and standardized method for the extraction and preparation of frog collagen peptides. Summary of the Invention

[0004] In view of the aforementioned problems, and in conjunction with the first aspect of the present invention, embodiments of the present invention provide a method for extracting and preparing collagen peptides from the forest frog, the method comprising: Fresh Northeast forest frogs are selected, the whole skin including the attached bones is peeled off, and non-target tissues such as fascia and fat are removed. The frogs are rinsed with clean water to remove blood and dirt, and then air-dried in an environment with a temperature of 25-30℃ and a relative humidity of ≤60% until the moisture content is ≤15%. The frogs are then screened through a 10-mesh standard sieve and the floating dust is blown away to obtain clean raw materials. The clean raw materials are pulverized at high speed and then passed through an 80-mesh standard sieve to obtain raw material powder with a particle size ≤180μm; The raw material powder is mixed with 15-20° aged Shaoxing wine at a mass ratio of 1:1.2-1.5 to form a uniform suspension system; Add neutral protease to the suspension system and enzymatically hydrolyze it at 48–50°C for 12–20 hours until the change rate of soluble protein content in the system is ≤5% / h, to obtain the enzymatic hydrolysate; The enzymatic hydrolysate was sequentially filtered through three layers, allowed to settle, and then filtered through a gradient filter to obtain the final product of forest frog collagen peptides.

[0005] Preferably, in step one, the air-drying process involves turning the plant over twice daily and avoiding direct sunlight. The dust is blown away using a high-speed blower with a wind speed of level 3.

[0006] Preferably, in step two, a high-speed universal pulverizer is used for pulverization at a speed of 2800 r / min, pulverizing 3 times for 30 seconds each time, with a 1-minute interval for cooling.

[0007] Preferably, in step three, the mass ratio of raw material powder to aged rice wine is 1:1.3, the stirring speed is 300 r / min, and the stirring time is 15 minutes.

[0008] Preferably, in step four, the neutral protease has an enzyme activity of 100,000 U / g and is added at 0.5% of the raw material mass. Stir every 2 hours for 3 minutes each time during enzymatic hydrolysis at a speed of 200 rpm.

[0009] Preferably, in step four, the enzymatic hydrolysis temperature is 49°C and the enzymatic hydrolysis time is 16 hours.

[0010] Preferably, in step five, the three-layer filtration consists of an 80-mesh double-layer gauze, a medium-speed qualitative filter paper, and a 0.45μm polyethersulfone filter membrane, arranged from top to bottom, and is performed using vacuum filtration with a vacuum degree of -0.08 to -0.09 MPa.

[0011] Preferably, in step five, the conditions for settling are a temperature of 20-25°C and a settling time of 24 hours. After settling, 90% of the clear liquid in the upper layer is extracted by siphoning.

[0012] Preferably, in step five, the gradient filter uses a three-layer gradient filter with pore sizes of 200 mesh, 300 mesh and 500 mesh respectively, and the extraction speed is controlled at 5-10 mL / min.

[0013] Preferably, the obtained frog collagen peptide product is a pale yellow to amber mucus with a peptide content ≥85%, moisture ≤10%, and heavy metals lead ≤0.5mg / kg and mercury ≤0.1mg / kg.

[0014] Based on the above, the enzymatic hydrolysis efficiency of frog skin collagen is significantly improved through standardized raw material pretreatment, precise crushing and synergistic soaking in rice wine. Combined with mild and precise temperature-controlled enzymatic hydrolysis using neutral protease, the natural activity of collagen peptides can be preserved to the greatest extent. The hydrolysis endpoint is highly controllable, avoiding over-hydrolysis and solving the problems of insufficient hydrolysis and low raw material utilization in traditional processes.

[0015] The process employs a three-layer vacuum filtration, static sedimentation, and gradient filter multi-stage purification method, which can thoroughly remove macromolecular impurities, colloids, and micro-residues. The resulting collagen peptide product has high purity, meets heavy metal residue standards, and has stable and controllable physicochemical indicators. It also exhibits good batch-to-batch quality consistency. The process is mild and easy to standardize, meeting the core requirements for product safety and stability in large-scale production. Attached Figure Description

[0016] Figure 1This is a schematic diagram of the execution flow of the method for extracting and preparing collagen peptides from forest frogs provided in this embodiment of the invention. Detailed Implementation

[0017] The present invention will now be described in detail with reference to the accompanying drawings. Figure 1 This is a schematic flowchart of a method for extracting and preparing collagen peptides from forest frogs according to an embodiment of the present invention. The following is a detailed description of the method for extracting and preparing collagen peptides from forest frogs.

[0018] Raw material pretreatment; Raw material selection: Select live Northeast forest frogs, peel off the whole skin (including attached bones), remove non-target tissues such as fascia and fat, and rinse with clean water 3 times to remove surface bloodstains; Air drying: Lay the washed frog skin flat on a ventilated drying rack and control the environmental conditions as follows: temperature 25-30℃, relative humidity ≤60%, natural ventilation, air dry until the moisture content is ≤15% (about 3-5 days), turn it over twice a day during the period, and avoid direct sunlight; Screening and purification: Use a 10-mesh standard sieve to screen the air-dried raw materials to remove moldy, insect-infested, and impurity-containing individuals. Then use a high-speed blower (speed 3) to blow away surface dust to obtain clean raw materials for use.

[0019] Pulverization process: The pretreated dry raw material is put into a high-speed universal pulverizer (model FW100, power 1.1kW), the speed is set to 2800r / min, pulverized 3 times, 30 seconds each time, cooled with a 1-minute interval, and passed through an 80-mesh standard sieve to collect the powder (particle size ≤180μm). Ingredient mixing: Take a 500mL high-temperature resistant borosilicate glass beaker (model GG-17), accurately weigh 100g of the pulverized raw material, add 120-150g of 15-20° aged Shaoxing wine (the mass ratio of raw material to Shaoxing wine is 1:1.2-1.5, preferably 1:1.3), place it on a magnetic stirrer (model HJ-3), and stir at 300r / min for 15 minutes to fully soak and mix the raw material and Shaoxing wine to form a uniform suspension system.

[0020] Enzyme addition: Add 0.5g of neutral protease (enzyme activity 100,000 U / g) (0.5% of the raw material mass) to the above suspension system and continue stirring for 5 minutes until the enzyme is completely dissolved; Enzymatic hydrolysis conditions control: Place the beaker in a CNC constant temperature water bath (model HH-S6), set the temperature to 48~50℃ (accuracy ±0.5℃), keep the water bath depth submerging 2 / 3 of the liquid surface in the beaker, and the enzymatic hydrolysis time is 12~20 hours (preferably 16 hours). Stir once every 2 hours (3 minutes each time, speed 200r / min) to ensure uniform enzymatic hydrolysis. Extraction endpoint determination: Samples were taken in the later stage of enzymatic hydrolysis, and the soluble protein content in the system was detected by the biuret method. When the protein content no longer increased significantly (change rate ≤ 5% / h), the extraction was determined to be complete.

[0021] Filtration system setup: A three-layer filtration device is used, consisting of, from top to bottom: 80-mesh double-layer gauze, medium-speed qualitative filter paper (15cm in diameter), and 0.45μm polyethersulfone filter membrane, assembled on a vacuum filtration flask (1L in volume). Vacuum filtration: Cool the enzymatic hydrolysate to room temperature (25°C), pour it into the filter device, start the vacuum pump (model 2XZ-1, vacuum degree -0.08~-0.09MPa), maintain a uniform filtration speed, collect the filtrate, and discard the solid residue (bone fragments, undigested protein). Filter membrane cleaning: Rinse the filter membrane with 50 mL of distilled water once after filtering 500 mL of enzymatic hydrolysate to avoid clogging and affecting filtration efficiency.

[0022] Settling container: Use a 1L sterile glass container, transfer the filtered extract into the container, and seal the container opening (leaving 1 / 10 of the space empty). Settling conditions: Place in a cool, clean environment (temperature 20-25℃, avoid vibration) and let it settle and purify for 24 hours to allow large molecular impurities and colloidal particles to settle to the bottom of the container. Supernatant separation: After settling, the upper 90% of the clear liquid is slowly extracted using a siphon method to avoid disturbing the sediment at the bottom.

[0023] Purification equipment: A small vacuum wine extractor (model YZ-50, pumping speed 50L / min) is used, equipped with a three-layer gradient filter (pore sizes of 200 mesh, 300 mesh, and 500 mesh respectively). Gradient filtration: Slowly pour the clear liquid obtained by siphoning into the feed inlet of the wine extraction equipment, control the extraction speed to 5-10 mL / min, and further remove tiny impurities through multiple layers of filter screen; Finished product collection: The purified mucous liquid is collected, which is the finished product of forest frog collagen peptides. The detection indicators are as follows: Appearance: Pale yellow to amber viscous liquid, odorless; Peptide content ≥85% (detected using the Folin-phenol method); Moisture content ≤10% (Karl Fischer process); Heavy metal content: lead ≤ 0.5 mg / kg, mercury ≤ 0.1 mg / kg; Storage conditions: The finished product should be packaged into sterile brown glass bottles, sealed, and stored in a cool, dry place (temperature ≤25℃, relative humidity ≤60%). Shelf life is 12 months. Example 1

[0024] Raw material dosage: 100g of dried whole frog skin, 130g of rice wine (ratio 1:1.3). Enzymatic hydrolysis conditions: 0.5g neutral protease, 49℃, 16 hours; Product yield: 82g of frog collagen peptides were obtained, with a peptide content of 88.3% and a moisture content of 8.5%, which meets the above quality standards. Example 2

[0025] Raw material dosage: 200g of dried whole frog skin, 240g of rice wine (ratio 1:1.2). Enzymatic hydrolysis conditions: 1.0 g neutral protease, 50℃, 14 hours; Product yield: 160g of frog collagen peptides were obtained, with a peptide content of 86.7% and a moisture content of 9.2%, which meets the above quality standards.

[0026] It should be noted that, in order to simplify the description of the present invention and thus help to understand one or more embodiments of the invention, multiple features may sometimes be grouped into one embodiment, drawing or description thereof in the foregoing description of the embodiments of the present invention.

[0027] The embodiments of this application have been described above with reference to the accompanying drawings. Unless otherwise specified, the embodiments and features in the embodiments of this application can be combined with each other. This application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of this application.

Claims

1. A method for extracting and preparing collagen peptides from the forest frog, characterized in that: Includes the following steps: Fresh Northeast forest frogs are selected, the whole skin including the attached bones is peeled off, and non-target tissues such as fascia and fat are removed. The frogs are rinsed with clean water to remove blood and dirt, and then air-dried in an environment with a temperature of 25-30℃ and a relative humidity of ≤60% until the moisture content is ≤15%. The frogs are then screened through a 10-mesh standard sieve and the floating dust is blown away to obtain clean raw materials. The clean raw materials are pulverized at high speed and then passed through an 80-mesh standard sieve to obtain raw material powder with a particle size ≤180μm; The raw material powder is mixed with 15-20° aged Shaoxing wine at a mass ratio of 1:1.2-1.5 to form a uniform suspension system; Add neutral protease to the suspension system and enzymatically hydrolyze it at 48–50°C for 12–20 hours until the change rate of soluble protein content in the system is ≤5% / h, to obtain the enzymatic hydrolysate; The enzymatic hydrolysate was sequentially filtered through three layers, allowed to settle, and then filtered through a gradient filter to obtain the final product of forest frog collagen peptides.

2. The method for extracting and preparing collagen peptides from the forest frog according to claim 1, characterized in that: In step one, the leaves should be turned over twice a day during the air-drying process, and direct sunlight should be avoided. The dust is blown away using a high-speed blower with a wind speed of level 3.

3. The method for extracting and preparing collagen peptides from the forest frog according to claim 1, characterized in that: In step two, a high-speed universal pulverizer is used for pulverization at a speed of 2800 r / min, pulverizing 3 times for 30 seconds each time, with a 1-minute interval for cooling.

4. The method for extracting and preparing collagen peptides from the forest frog according to claim 1, characterized in that: In step three, the mass ratio of raw material powder to aged rice wine is 1:1.3, the stirring speed is 300 r / min, and the stirring time is 15 minutes.

5. The method for extracting and preparing collagen peptides from the forest frog according to claim 1, characterized in that: In step four, the neutral protease has an enzyme activity of 100,000 U / g and is added at 0.5% of the raw material mass. Stir every 2 hours for 3 minutes each time during enzymatic hydrolysis at a speed of 200 rpm.

6. The method for extracting and preparing collagen peptides from the forest frog according to claim 1, characterized in that: In step four, the enzymatic hydrolysis temperature is 49℃ and the enzymatic hydrolysis time is 16 hours.

7. The method for extracting and preparing collagen peptides from the forest frog according to claim 1, characterized in that: In step five, the three-layer filtration, from top to bottom, consists of 80-mesh double-layer gauze, medium-speed qualitative filter paper, and 0.45μm polyethersulfone filter membrane, using vacuum filtration with a vacuum degree of -0.08 to -0.09MPa.

8. The method for extracting and preparing collagen peptides from the forest frog according to claim 1, characterized in that: In step five, the conditions for settling are a temperature of 20-25°C and a settling time of 24 hours. After settling, 90% of the clear liquid in the upper layer is extracted using a siphon method.

9. The method for extracting and preparing collagen peptides from the forest frog according to claim 1, characterized in that: In step five, the gradient filter uses a three-layer gradient filter with pore sizes of 200 mesh, 300 mesh and 500 mesh, and the extraction speed is controlled at 5-10 mL / min.

10. The method for extracting and preparing collagen peptides from the forest frog according to claim 1, characterized in that: The obtained frog collagen peptide product is a pale yellow to amber mucus with a peptide content of ≥85%, moisture content of ≤10%, and heavy metal content of ≤0.5mg / kg lead and ≤0.1mg / kg mercury.

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